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胰高血糖素样肽1对骨骼肌成肌细胞增殖的调控及信号机制 被引量：3

Regulatory effect of glucagon-like peptide-1 on cell proliferation of skeletal myoblast strain L6 and its possible signal mechanism
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摘要 目的探讨胰高血糖素样肽1（GLP-1）对骨骼肌成肌细胞增殖的调控作用及其信号机制。方法体外培养大鼠骨骼肌成肌细胞株L6，并按随机数字表法分为4组：（1）对照组，培养液中除常规成分外不再添加其他物质；（2）GLP-1组，培养液中加入终浓度10nmol／L的GLP-1；（3）磷脂酰肌醇3激酶（PI3K）抑制剂组，培养液中加入终浓度为50nmol／L的PI3K特异性抑制剂渥曼青霉素；（4）GLP-1＋PI3K抑制剂组，培养液中加入终浓度10nmol／L的GLP-1和终浓度50nmol／L的渥曼青霉素。各组细胞接受上述处理后分别继续培养24、48、72h，采用噻唑蓝法测定细胞增殖活性（结果用吸光度值表示）；继续培养24h时，用流式细胞仪检测细胞周期分布，行免疫组织化学染色检测细胞中增殖细胞核抗原（PCNA）表达，蛋白质印迹法检测磷酸化（P-）蛋白激酶B（Akt）、p-PI3K的蛋白表达水平。对实验数据行方差分析。结果（1）GLP-1组细胞接受处理后48、72h，增殖活性分别为0．660±0．120、0．870±0．240，均显著高于对照组（0．530±0．060、0．700±0．100，F值分别为5．46、5．90，P〈0．05或P〈0．01）。PI3K抑制剂组各时相点增殖活性均低于对照组。GLP-1＋PI3K抑制剂组处理48、72h，细胞增殖活性分别为0．510±0．080、0．740±0．160，与GLP-1组比较差异均有统计学意义（F值分别为5．46、5．90，P〈0．05或P〈0．01）。（2）处理后24h，GLP-1组S期细胞百分比为（15．7±0．4）％，显著高于对照组[（13．6±0．6）％]和GLP-1＋PI3K抑制剂组[（10．1±0．6）％]；PI3K抑制剂组s期细胞百分比为（6．84±1．2）％，明显低于对照组。以上各项比较F值均为15．39，P值均小于0．01。（3）处理后24h，GLP-1组细胞PCNA增殖指数为（51．24±1．18）％，显著高于对照组[（36．72±1．56）％]和GLP-1＋PI3K抑制剂组[（25．90±1．22）％]；PI3K抑制剂组PCNA增殖指数为（21．70±0．09）％，明显低于对照组。以上各项比较F值均为783．80，P值均小于0．05。（4）处理后24h，GLP-1组细胞p-Akt蛋白表达水平显著高于其余3组，PI3K抑制剂组明显低于对照组。以上各项比较F值均为94．43，P值均小于0．01。GLP-1组、CLP-1＋PI3K抑制剂组p-PI3K蛋白表达水平与对照组接近（F值均为20．94，P值均大于0．05），PI3K抑制剂组较对照组明显降低（F=20．94，P〈0．05）。结论GLP-1可直接作用于骨骼肌成肌细胞，通过加速细胞周期进程，增加DNA合成，促进细胞增殖。该作用与PI3K／Akt信号途径密切相关。 Objective To study the regulatory effect of glucagon-like peptide-1 （ GLP-1 ） on cell proliferation of skeletal myoblast strain L6 and its possible signal mechanism. Methods L6 cells cultured in DMEM high glucose culture medium containing 10% FBS were divided into control group （C, without addition）, GLP-1 group （G, added with 10 nmol/L GLP-1 ）, PI3K inhibitor group （W, added with 50 nmol/L PI3K specific inhibitor wortmannin）, and GLP-1 ＋ PI3K inhibitor group （ GW, added with 10 nmol/L GLP-1 and 50 nmol/L wortmannin） according to the random number table. Cell proliferation activity was detected with MTT assay at post culture hour （PCH） 24, 48, 72 （denoted as absorbance value）. At PCH 24, the change in cell cycle was evaluated with flow cytometer, the expression level of proliferating cell nuclear antigen （PCNA） was determined with immunohistochemical staining, the protein levels of phosphorylated PI3K （p-PI3K） and p-Akt were determined with Western blotting. Data were processed with multi-group analysis of variance. Results （1） The cell proliferation activity at PCH 48, 72 in G group was respectively 0. 660 ± 0. 120, 0. 870 ± 0. 240, all significantly higher than those in C group （0. 530 ± 0. 060, 0. 700 ± 0. 100, with F value respectively 5.46, 5.90, P 〈 0.05 or P 〈 0.01 ）. The cell proliferation activity in W group at each time point was lower than that in C group. The cell proliferation activity in GW group at PCH 48, 72 was respectively 0. 510 ± 0. 080, 0. 740 ± 0. 160, all lower than those in G group （withFvalue respectively 5.46, 5.90, P 〈0.05 or P 〈0.01）. （2） The percentage of S phase cell in G group at PCH 24 [ （15.7 ±0.4） % ] was significantly higher than that in C group [ （13.6 ±0.6） % ] and GW group [（10.1 ±0.6）%1, while that in W group [（6.8 ±1.2）%] was lower than that in C group （with Fvalues all equal to 15.39, P values all below 0.01）. （3） PCNA level in G group at PCH 24 [ （51.24 ± 1. 18）% ] was markedly higher than that in C group [ （36.72 ± 1.56）% ] and GW group [ （25.90 ± 1.22）% ], and while in W group [ （21.70 ±0.09）% ] was lower than that in C group （with F values equal to 783.80, P values all below 0.05）. （4） The protein level of p-Akt in G group at PCH 24 was significantly higher than that in the other 3 groups, while that in W group was lower than that in C group （ with F values equal to 94.43, P values all below 0.01 ）. There was no obvious difference in protein level of p-PI3K at PCH24 among G, GW, and C groups （ F =20.94,P 〉0.05）. The protein level ofp-PI3K at PCH24 in W group was lower than that in C group （ F =20.94, P 〈0.05）. Conclusions GLP-1 can promote cell proliferation of skeletal myoblast by accelerating the progression of cell cycle and increasing the synthesis of DNA, which can be attributed to PI3K/Akt signal pathway.
作者 申传安 柴家科 马丽 海恒林 张琳
机构地区 解放军总医院第一附属医院全军烧伤研究所
出处 《中华烧伤杂志》 CAS CSCD 北大核心 2011年第5期332-336,共5页 Chinese Journal of Burns
基金 基金项目：国家自然科学基金（30971128）
关键词 胰高血糖素样肽1 成肌细胞 骨骼肌 细胞增殖 信号通路 Glucagon-like peptide-1 Myoblasts, skeletal Cell proliferation Signal pathway
分类号 R363 [医药卫生—病理学]
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参考文献10

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共引文献20

同被引文献27

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中华烧伤杂志
2011年 第5期

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